This invention provides isolated nucleic acids of p-Hyde genes, proteins, analogs, fragments, mimetics, mutants, synthetics, and variants thereof of the p-Hyde family. This invention is directed to a method of inducing susceptibility to apoptosis with p-Hyde, a method of suppressing tumor growth with p-Hyde, and a method of treating a subject with cancer with p-Hyde alone or in combination with radiation, chemotherapy, or UV mimetic drugs.
Prostate cancer is the most common malignancy in men with over 317,000 new cases and the second leading cause of male caner deaths in the United States (Boring et al., 1993; Steiner et. al, 1995). The molecular mechanisms responsible for the development, progression, and metastasis of prostate cancer remain largely unknown. Up to 20% of prostate cancers occur in men under the age of 65 years of age (Silverberg, 1986) suggesting that prostate carcinogenesis is not only associated with aging, but also to hereditary factors (Silverberg, 1987; McLellan and Norman, 1995; Carter et al., 1992). Genetic linkage studies of 691 affected families have revealed that an earlier age of onset of the disease in the proband and the presence of multiple affected family members are important determinants that increase the risk of prostate cancer. The pattern of inheritance of the putative prostate cancer gene appears to be autosomal dominant with an 88% penetrance rate (Steinberg, 1990). Thus, hereditary factors play an important role in prostate oncogenesis.
Like many carcinomas, prostate cancer formation is a multistep process involving tumor initiation, promotion, conversion, and progression (Carter et al., 1990; Sandberg, 1992). This process is driven by chromosomal instability, spontaneous mutations, and carcinogen induced genetic and epigenetic changes. Chromosomal instability leads to the total or partial gain or loss of chromosomes, translocations, and other abnormalities. Spontaneous mechanisms are age-related and include activation of oncogenes or inactivation of tumor suppressor genes by genetic mutations. These mutations result in the misincorporation of nucleotides during DNA replication of the coding region, alteration of the intron-exon junction sequences affecting the splicing mechanism, and aberrations of regulatory sequences changing the control of critical genes. These mutations escape genetic surveillance by a battery of DNA repair mechanisms and its associated gene products, such as p53 (Effert et al., 1992, Isaacs et al, 1991; Mellon et al., 1992) and p21 (El-Deiry et al., 1994) and PCNA (Templeton et al., 1996). Carcinogen-induced genetic ad epigenetic changes initiate tumors as a consequence of the direct damaging effects of carcinogenic agents of the DNA altering gene expression. Tumor initiation is subsequently followed by tumor promotion as affected cells have selective reproductive and clonal expansion capabilities through altered signaling transduction and proliferation responses to growth factors, resistance to cytotoxicity, and deregulation of terminal differentiation (Yuspa and Poirier, 1988; Weinstein, 1987). Finally, tumor promotion is succeeded by other genetic mutational events that lead to loss of hormone sensitivity, increased cell motility, invasion, alterations in programmed cell death and metastasis. Accordingly, the initiation and progression of cancer is a multistep process whereby genetic alterations or mutations of critical genes ultimately dictate defined cell phenotypes which differ in regard to many important cellular activities including cell proliferation, differentiation, and programmed cell death. The exact mutational events responsible for the multistep progression of prostate cancer, however, is unknown. A better understanding of the molecular mechanisms responsible for prostate cancer may lead to new therapies to combat, and perhaps, to even prevent prostate cancer.
The present invention provides isolated nucleic acids encoding P-Hyde genes of the p-Hyde family. The p-Hyde gene as shown herein is associated with: (1) the regression of tumor growth in vivo (2) the induction to susceptibility to apoptosis caused by UV or chemotherapy induced DNA damage, and (3) prevention of DNA repair with the upregulation of apoptosis as the result of UV damage and the failure to repair DNA.
This invention provides a novel class of genes which act as inhibitor of a DNA repair enzyme and induce susceptibility of cancer cells to cell death. Also, this invention provides isolated nucleic acids which encodes a mammalian p-Hyde protein which induce susceptibility of a cancer cell to cell death, including allelic, analogs, fragments, mimetics, mutants, synthetics, or variants thereof. This invention provides an isolated nucleic acids which encodes a human p-Hyde protein which induces susceptibility of a cancer cell to cell death, including allelic, analogs, fragments, mimetics, mutants, synthetics, or variants thereof
Within this invention is provided a nucleic acid which has the nucleotide sequence as shown SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO:9.
Also, within the invention is a nucleic acid molecule which encodes a fragment of a polypeptide having the amino acid sequence of SEQ ID NO: 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8 or SEQ ID NO: 10, the fragment including at least 15 (25, 30, 50, 60, or 63) contiguous amino acids of SEQ ID NO: 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8 or SEQ ID NO 10.
Also, wit the invention is a nucleic acid molecule having the nucleotide sequence which is at least about 82%, 84%, 85%, 87%, 90%, 92%, 95%, or 98% identical to the nucleic acid sequence as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO: 9.
Also, within this invention is a nucleic acid molecule which encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, SEQ ID NO 4, SEQ ID NO 6, SEQ ID NO 8, or SEQ ID NO 10 wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO: 2, SEQ ID NO 4, SEQ ID NO6, SEQ ID NO 8 or SEQ ID NO 10 or the complement thereof under stringent conditions.
Also within the invention are isolated p-Hyde proteins having an amino acid sequence that is at least about 82%, 84%, 85%, 87%, 90%, 95%, or 98% identical to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO 9.
Also within the invention are: an isolated p-Hyde protein protein which is encoded by a nucleic acid molecule having a nucleotide sequence that is at east about 65%, preferably 75%, 80%, 85%, or 95% identical to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO 9; and an isolated p-Hyde protein protein which is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:5, SEQ ID NO:7 or SEQ ID NO 9, or the complement thereof.
The p-Hyde protein of the present invention, or biologically active portions thereof, can be operably linked to a non-p-Hyde polypeptide (e.g., heterologous amino acid sequences) to form p-Hyde fusion proteins.
This invention provides a vector comprising the isolated nucleic acid encoding P-Hyde gene. This invention provides a replication-defective recombinant E1/E3 deleted adenovirus containing a truncated RSV promoter and the P-Hyde cDNA gene (AdRSVpHyde).
This invention provides an oligonucleotide of at least 15 nucleotides capable of specifically hybridizing with a sequence of nucleotides present within a nucleic acid which encodes the p-Hyde, or a sequence which is complementary to the nucleic acid which encodes the p-Hyde. This invention provides au antisense molecule, triplex oligonucleotide, or ribozyme which is capable of specifically hybridizing with the isolated nucleic acid encoding p-Hyde.
This invention provides a method for producing a polypeptide which comprises growing the host vector system under suitable conditions permitting production of the polypeptide and recovering the polypeptide so produced. In one embodiment the method of obtaining a polypeptide in purified form comprises: (a) introducing the vector into a suitable host cell; (b) culturing the resulting cell so as to produce the polypeptide; (c) recovering the polypeptide produced in step (b); and (d) purifying the polypeptide so recovered.
This invention provides a polypeptide comprising the amino acid sequence of a p-Hyde. This invention provides a fusion protein or chimeric comprising the polypeptide. This invention provides an antibody which specifically binds to the polypeptide. This invention provides a pharmaceutical composition comprising an amount of the polypeptide and a pharmaceutically effective carrier or diluent.
This invention provides a method for determining whether a subject carries a mutation in the p-Hyde gene which comprises: (a) obtaining an appropriate nucleic acid sample from the subject; and (b) determining whether the nucleic acid sample from step (a) is, or is derived from, a nucleic acid which encodes mutant p-Hyde so as to thereby determine whether a subject carries a mutation in the p-Hyde gene. In one embodiment is the nucleic acid sample in step (a) comprises mRNA corresponding to the transcript of DNA encoding a mutant p-Hyde, and wherein the determining of step (b) comprises: (i) contacting the mRNA with the oligonucleotide under conditions permitting binding of the mRNA to the oligonucleotide so as to form a complex; (ii) isolating the complex so formed; and (iii) identifying the mRNA in the isolated complex so as to thereby determine whether the mRNA is, or is derived from, a nucleic acid which encodes mutant p-Hyde.
This invention provides a method for screening a tumor sample from a human subject for a somatic alteration in a p-Hyde gene in said tumor which comprises gene comparing a first sequence selected form the group consisting of a p-Hyde gene from said tumor sample, p-Hyde RNA from said tumor sample and p-Hyde cDNA made from mRNA from said tumor sample with a second sequence selected from the group consisting of p-Hyde gene from a nontumor sample of said subject, p-Hyde RNA from said nontumor sample and p-Hyde cDNA made from mRNA from said nontumor sample, wherein a difference in the sequence of the p-Hyde gene, p-Hyde RNA or p-Hyde cDNA from said tumor sample from the sequence of the p-Hyde gene, p-Hyde RNA or p-Hyde cDNA from said nontumor sample indicates a somatic alteration in the p-Hyde gene in said tumor sample.
This invention provides a method for screening a tumor sample from a human subject for the presence of a somatic alteration in a p-Hyde gene in said tumor which comprises comparing p-Hyde polypeptide from said tumor sample from said subject to p-Hyde polypeptide from a nontumor sample from said subject to analyze for a difference between the polypeptides, wherein said comparing is performed by (i) detecting either a full length polypeptide or a truncated polypeptide in each sample or (ii) contacting an antibody which specifically binds to either an epitope of an altered p-Hyde polypeptide or an epitope of a wild-type p-Hyde polypeptide to the p-Hyde polypeptide from each sample and detecting antibody binding, wherein a difference between the p-Hyde polypeptide from said tumor sample from the p-Hyde polypeptide from said nontumor sample indicates the presence of a somatic alteration in the p-Hyde gene in said tumor sample.
This invention provides a method for identifying a chemical compound which is capable inducing susceptibility to cell death which comprises: (a) contacting the p-Hyde with a chemical compound under conditions permitting binding between the p-Hyde and the chemical compound; (b) detecting specific binding of the chemical compound to the p-Hyde; and (c) determining whether the chemical compound inhibits the p-Hyde so as to identify a chemical compound which is capable of capable inducing susceptibility to cell death.
This invention provides a method of inhibiting the growth of cancer cells, comprising the steps of obtaining the cells and contacting the cells of the subject with a replication-deficient adenovirus type 5 expression vector comprising an adenovirus genome having a deletion in the E1 and E3 region of the genome and an insertion within the region of a nucleic acid encoding p-Hyde under the control of a Rous Sarcoma virus promoter, thereby inhibiting the growth of the prostate cancer cells.
This invention provides a method of inhibiting the growth a prostate cancer cells, comprising: 1) obtaining a sample of prostate cells from a subject; 2) contacting the cells with a replication deficient adenovirus type 5 expression vector which comprises an adenovirus genome having a deletion in the E1 and E3 regions of the genome and an insertion within the regions of a p-Hyde cDNA under the control of a Rous Sarcoma virus promoter; and 3) introducing the cells into the subject, thereby inhibiting the growth of the cancer cells.
This invention provides a method of suppressing the growth of cancer cells in a subject, comprising introducing into the cancer cell an amount of a nucleic acid encoding a p-Hyde protein, a nucleic acid encoding a fragment of p-Hyde protein, or the nucleic acid encoding a mutant p-Hyde protein, thereby suppressing the growth of cancer cells in the subject.
This invention provides a method of suppressing growth of cancer cells in a subject, comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a nucleic acid encoding a p-Hyde protein, a nucleic acid encoding a fragment of p-Hyde protein, or the nucleic acid encoding a mutant p-Hyde protein and a pharmaceutical acceptable carrier or diluent, thereby suppressing the growth of cancer cells in the subject.
This invention provides a method of inducing susceptibility to apoptosis of cancer cells in a subject, comprising introducing into the cancer cell an amount of a nucleic acid encoding a p-Hyde protein, a nucleic acid encoding a fragment of p-Hyde protein, or the nucleic acid encoding a mutant p-Hyde protein, thereby inducing susceptibility to apoptosis.
This invention provides a method of inducing susceptibility to apoptosis of cancer cells in a subject, comprising administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a nucleic acid encoding a p-Hyde protein, a nucleic acid encoding a fragment of p-Hyde protein, or the nucleic acid encoding a mutant p-Hyde protein and a pharmaceutical acceptable carrier or diluent thereby inducing susceptibility to apoptosis.
This invention provides a method of treating a subject with cancer which comprises administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a nucleic acid encoding a p-Hyde protein, a nucleic acid encoding a fragment of p-Hyde protein, or the nucleic acid encoding a mutant p-Hyde protein and a pharmaceutical acceptable carrier or diluent, thereby treating the subject with cancer.
This invention provides a method of treating a subject with cancer, comprising: 1) administering to the subject a pharmaceutical composition comprising a therapeutically effective amount of a nucleic acid encoding a p-Hyde protein, a nucleic acid encoding a fragment of p-Hyde protein, or the nucleic acid encoding a mutant p-Hyde protein in combination with radiation, chemotherapy, or UV mimetic drugs; and 2) a pharmaceutical acceptable carrier or diluent, thereby treating the subject with cancer.
This invention a method of treating a subject with cancer, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising 1) an adenovirus type 5 expression vector which comprises a adenovirus genome having a deletion in the E1 and E3 regions of the genome and an insertion within the regions of a full length sense p-Hyde cDNA under the control of a Rous Sarcoma virus promoter, and 2) a suitable carrier or diluent, thereby treating the subject with cancer. In one embodiment the cancer is selected from a group consisting of melanoma; lymphoma; leukemia; and prostate, colorectal, pancreatic, breast, brain, or gastric carcinoma.
Lastly, the present invention provides the means necessary for production of gene-based therapies directed at cancer cells. These therapeutic agents may take the form of polynucleotides comprising all or a portion of the p-Hyde locus placed in appropriate vectors or delivered to target cells in more direct ways such that the function of the p-Hyde protein is reconstituted. Therapeutic agents may also take the form of polypeptides based on either a portion of, or the entire protein sequcnce of p-Hyde. These may functionally replace the activity of p-Hyde in vivo.